ES2464123T3 - Procedure for 2-sulphation of glycosides - Google Patents

Abstract

A method for sulfating an α-L-iduronate glycoside in position 2, comprising: a) treating an α-L-iduronate glycoside having hydroxyl groups in positions 2 and 4 in cis relation with a tin reagent, providing a α-L-iduronate 2,4-stanylene glycoside acetal; and b) treating the 2,4-stanylene acetal with a sulphating agent, providing a glycoside of α-L-iduronate 2- sulfated.

Description

Procedure for 2-sulphation of glycosides

5 Background of the invention

[0001] The development of the new technology for the neonatal screening of Hunter syndrome (mucopolysaccharidosis II) is guaranteed due to the development of treatments that are very effective when initiated early in life. This lysosomal storage disease is caused by the deficiency of the enzyme iduronate-2-sulfatase, which is necessary for the degradation of dermatan sulfate and heparan sulfate, two components of cellular glucosaminoglycans. The testing of this sulfatase requires the use of α-L-iduronate glycosides that contain a 2-position sulfate.

[0002] Synthetic substrates used to test iduronate-2-sulfatase in vitro are usually

15 sulfated disaccharides derived from degradation with nitrous acid of heparin. Such substrates have been useful for the development of a tandem mass spectrometry assay for neonatal screening of Hunter syndrome. However, more recently it has been shown that large-scale synthesis using nitrous acid degradation of heparin is impracticable to obtain the amount of material needed to support global neonatal screening for Hunter syndrome.

[0003] Abad-Romero, Beatriz, et al., 2009 describes methyl xylobioside and methyl xylotioside prepared from peracetylated anomeric xylosyl trichloroacetamidates by reaction with methanol followed by Zemplen deacetylation.

[0004] US 5,874,548 A describes a direct procedure for the regioselective sulphation of an organic molecule having hydroxyl groups optionally derivatized into at least two adjacent carbon atoms.

[0005] US 5,783,693 A discloses sulfated disaccharides characterized by the ability to inhibit the binding of selectin to its physiologically relevant ligand

[0006] US 6,184,196 B1 describes the synthesis of sulfonated sucrose compounds, surfactants and intermediate cyclic sulfates.

[0007] Davis, B. G., et al, 1999 reviews the synthesis and application of glucoconjugates and Gridley, J. J., et al., 2000 describes the construction of �-D-mannose and �-D-mannosamine linkages.

[0008] There is a need for a new procedure for the total synthesis of appropriate substrates that can be used on a scale of tens of grams per year.

SUMMARY OF THE INVENTION

[0009] The present invention provides a method for sulfating a glycoside at position 2. In one embodiment, the method includes treating a glycoside having hydroxyl groups at positions 2 and 4 in

Cis ratio with a tin reagent, providing a 2,4-stanylene acetal glycoside, and treating the 2,4-stanylene acetal with a sulphating agent, providing a 2-sulfated glycoside. The process of the invention selectively sulfates position 2 of the glycoside preferably to position 4. In one embodiment, the 2-sulfated glycoside is sulfated in position 2 with a selectivity greater than about 90% (with respect to sulfation in position 4 ). In one embodiment, the 2-sulfated glycoside is sulfated in position 2 with a selectivity that is greater than about 95% (with respect to sulfation in position 4).

[0010] In one embodiment, the glycoside is iduronate glycoside. In one embodiment, the glycoside is an α-L-iduronate glycoside.

[0011] Tin reagents useful in the process of the invention include tin reagents capable of forming stanylene acetals. Representative tin reagents include dialkyltin (IV) oxides such as dibutyltin (IV) oxide.

[0012] Sulfation agents useful in the process of the invention include sulfur trioxide reagents such as sulfur trioxide complexes with trimethylamine, pyridine complexes and sulfur trioxide with N, N-dimethylformamide.

DESCRIPTION OF THE DRAWINGS

[0013] The above aspects and many of the accompanying advantages of this invention will be more readily appreciated and will be better understood by reference to the following detailed description, when taken together with the accompanying drawings, in which:

FIGURE 1 is a schematic illustration of the selective 2-sulphation of a representative iduronate glycoside according to the process of the invention;

FIGURE 2 is a schematic illustration of the synthesis of an iduronate glycoside starting material (aL-iduronate glycoside methyl ester) useful in the 2-sulphation of a representative iduronate glycoside according to the process of the invention. ; Y

FIGURE 3 is a schematic illustration of the selective 2-sulphation of a generalized iduronate glycoside according to the process of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides a method for sulfating an aL-iduronate glycoside at position 2. In one embodiment, the method includes treating said glycoside with a tin reagent, providing a 2,4-stanylene glycoside acetal , and treating the 2,4-stanylene acetal with a sulphating agent, providing a 2-sulfated glycoside.

[0015] The nature of the glycoside aglycone is not critical in the process of the invention. Suitable glycons do not interfere with the sulfation of the procedure. Representative glycones include aromatic compounds (for example, compounds containing phenyl or benzyl linked through the aromatic group, for example, coumarins), other carbohydrates and polycyclic compounds (for example, steroids).

[0016] The process of the invention selectively sulfates position 2 of the glycoside preferably to position 4. In one embodiment, the 2-sulfated glycoside is sulfated in position 2 with a selectivity greater than about 90% (relative to sulfation in position 4). In one embodiment, sulfate glucoside 2 is sulfated in position 2 with a selectivity greater than about 95% (based on sulphation in position 4).

[0017] As noted above, the nature of the glycoside aglycone can vary widely. In one embodiment, the glycoside is an iduronate glycoside. The a-L-iduronate glycoside sulfates formed by the process of the invention are useful in tests of the iduronate-2-sulfatase enzyme in the neonatal screening of Hunter syndrome (mucopolysaccharidosis II).

[0018] In one embodiment, the glycoside is an iduronate glycoside having the formula:

40 in which the glycoside forms an original ion when subjected to tandem mass spectrometry with electrospray ionization, M is a residue that is cleaved from the original ion, providing a fragmented ion by collision-induced dissociation, L covalently binds M with the glycoside moiety and R is a C1-C6 alkyl group. Representative M groups include butyloxycarbonyl (M is C4H9OC (= O) -). Representative L groups include linkages such as -NH- (CH2) n-NHC (= O) CH2-, in which n is 1-8. Sulfation of this iduronate glycoside

45 provides a 2-sulfated glycoside having the formula:

in which M, L and R are as before and X is a hydrogen or a counterion. Representative X groups include ammonium ions such as trimethylammonium ion and metal ions such as sodium ion. Saponification of this 2-sulfated iduronate glycoside provides an iduronate glycoside having the formula:

10 in which M, L and X are as before. [0019] In another embodiment, the glycoside is an α-L-iduronate glycoside having the formula:

Sulfation of this iduronate glycoside provides a 2-sulfated glycoside having the formula:

Saponification of this 2-sulfated iduronate glycoside provides an iduronate glycoside having the formula:

5 Saponified 2-sulfated iduronate glycoside is useful in iduronate-2-sulfatase enzyme assays as described below.

[0020] Tin reagents useful in the process of the invention include tin reagents

10 capable of forming stanylene acetals. Representative tin reagents include dialkyltin (VI) oxides such as dibutyltin (IV) oxide.

[0021] Sulfation agents useful in the process of the invention include sulfur trioxide reagents such as sulfur trioxide complexes with trimethylamine, pyridine complex and sulfur trioxide with

15 N, N-dimethylformamide.

[0022] As noted above, in one embodiment the present invention provides a method for the selective 2-sulphation of iduronate glycosides. Although there are a number of reports of the synthesis of sulfated saccharide building blocks that have been used to prepare heparin fragments and

20 heparan sulfate, there are no reports on the easy incorporation of sulfate to position 2 of a-Liduronate glycosides. In the process of the invention, the iduronate glycosides are sulphated in position 2 with a selectivity greater than 90% compared to sulphation in position 4.

[0023] The 2-sulfated compound can be used to test iduronate-2-sulfatase using an assay

25 fluorimetric or by tandem mass spectrometry with electrospray ionization. The fluorimetric assay is made possible by the presence of the umbelliferyl moiety. In this case, the test mixture is supplemented with the enzyme a-L-iduronidase, which cleaves the glycosidic linkage releasing the fluorescent umbelliferone only after the iduronate-2-sulfatase removes the 2-sulfate group. For the tandem mass spectrometry test, the coupling enzyme a-L-iduronidase is not necessary. In this case, a-L glycoside is detected

30 iduronate directly desulphated by tandem mass spectrometry. The presence of the butyloxycarbonyl group (BOC) directs the stability of the original ion so that fragmentation proceeds exclusively by carbamate cleavage (loss of 100 Da).

[0024] In one embodiment, the invention provides a three-stage process for sulfate introduction.

35 at position 2 starting from an ether of iduronate glycosides (for example, an α-L-iduronate glycoside). The process involves the protection of the 2-and 4-hydroxyl groups of the iduronate moiety such as dibutylmethanylene acetal, the selective sulfation with sulfur trimethylamine trioxide and the deprotection of the ester, providing the desired 2-sulfate.

[0025] A schematic illustration of the selective 2-sulphation of a representative iduronate glycoside (specifically, aL-iduronate glycoside) according to the process of the invention is described in Example 2, and is illustrated in FIGURE 1 With respect to FIGURE 1, the preparation begins with the glycoside methyl ester of aL-iduronate 9b, prepared as described in example 1 and schematically illustrated in FIGURE 2. Treatment of methyl ester 9b with 1.5 equivalents of dibutyltin oxide in anhydrous methanol a

45 reflux protects the 2- and 4-hydroxyl groups as stanylene acetal. Acetal was used without further purification. The acetal was dissolved in anhydrous N, N-dimethylformamide and treated with 1.5 equivalents of sulfur trimethylamine trioxide complex for 24 h at 55 ° C. The crude product was subjected to cation exchange chromatography to convert the trimethylammonium salt of the sulfate into the sodium salt, which was purified by flash chromatography on silica gel to provide the 2-sulfate methyl ester 10. The methyl ether was solubilized 2 -Sulfated 10 in

50 methanol / water and treated with incremental amounts of aqueous sodium hydroxide to saponify the methyl ester. The crude product was purified by flash chromatography on silica gel, giving glycoside 2 sulfate 11 with 96% purity (61% overall yield of compound 9b). The structure was confirmed by 1 H-NMR and mass spectrometry with electrospray ionization. Sulfation selectivity was shown by 1 H-NMR analysis, which indicated that 96% of the product is sulfated in position 2 and 4% in

position 4.

[0026] Because there is no known enzyme capable of hydrolyzing sulfate in position 4, the removal of trace amounts of 4-sulfate is not necessary before the enzymatic assay of Hunter syndrome. The method of the invention provides the iduronate-2-sulfatase substrate 11 useful in the neonatal screening test for Hunter syndrome.

[0027] The following examples are provided in order to illustrate, and not limit, the invention.

Examples

General procedures

[0028] The reactions were carried out in dry solvents in oven-dried glass containers in

15 atmosphere of N2. Thin layer chromatography was carried out on silica plates (silica gel 60, F-254 (0.25 mm)). The chemical shifts of 1 H-NMR are reported in parts per million (5) using the methanol peak as internal standard (3.31 ppm). Mass spectra with electrospray ionization were acquired on a Bruker Esquire LC00066 ion trap spectrometer. Flash chromatography was carried out with silica gel (40-63 µm).

Example 1

Preparation of a-L-iduronate glycoside methyl ester

[0029] The preparation of the a-L-iduronate 9b glycoside methyl ester is described below and is illustrated in FIGURE 2.

[0030] (2,3,4-Tri-O-acetyl-D-glucopyranosylfluoride) methyl uronate (2). Methyl 1,2,3,4-tetra-O-acetyl, -D-glucopyranosyluronate 1 (4.98 g, 13.25 mmol, 1 eq) was suspended at 0 ° C in 67 ml of 33% hydrobromic acid in acetic acid under nitrogen atmosphere. After stirring for 15 min at 0 ° C, the reaction mixture was allowed to warm to room temperature and stirred for 2 h. The reaction mixture was then diluted with toluene and concentrated in vacuo. The residue was diluted with 250 ml of ethyl acetate and washed with 150 ml of cold saturated sodium bicarbonate and 150 ml of cold brine. The organic phase was dried over MgSO4 and concentrated in vacuo to provide the crude bromide derivative used directly in the next step. Intermediate dissolved

35 bromide in 167 ml of anhydrous acetonitrile under a nitrogen atmosphere at room temperature. Silver fluoride (3.36 g, 26.49 mmol, 2 eq) was then added. The reaction mixture was stirred for a total of 21 h in the dark. The reaction mixture was filtered through Celite and the filtrate was concentrated in vacuo. Column chromatography on silica gel (hexane: AcOEt 4: 1 to 2: 1) provided product 2 (3.3 g, 74%).

[0031] (5-Bromo-2,3,4-tri-O-acetyl-D-glucopyranosylfluoride) methyl uronate (3). A suspension of 2 (3.3 g, 9.8 mmol, 1 eq) and N-bromosuccinimide (3.32 g, 18.65 mmol, 1.9 eq) was stirred in anhydrous carbon tetrachloride under a nitrogen atmosphere and reflux with irradiation for a total of 6 h. N-Bromosuccinimide (3.32 g, 18.65 mmol, 1.9 eq) was added after 2 and 4 h of reaction. The reaction mixture was cooled to room temperature and filtered through glass wool. The solvent was removed in vacuo. Column chromatography on silica gel

45 (hexane: AcOEt 3: 1) provided product 3 (3.12 g, 77%).

[0032] (2,3,4-Tri-O-acetyl-a-L-idopyranosylfluoride) methyl uronate (4). The bromide 3 (3.16 g, 7.61 mmol, 1 eq) was dissolved in 50 ml of anhydrous benzene and stirred under a nitrogen atmosphere. Tributyltin hydride (3.1 ml, 11.4 mmol, 1.5 eq) was added and the reaction mixture was heated at reflux for 40 min. The mixture was cooled to room temperature and the solvent was removed in vacuo. Column chromatography on silica gel (toluene: AcOEt 8: 1 to 6: 1) provided product 4 (1.67 g, 65%).

[0033] 7-Acetoxicumarin-4-acetate (2 ’, 2’, 2’-trichlorethyl) (6a). 2,2,2-Trichloroethanol (431 µL, 4.5 mmol, 1.25 eq) was added to a suspension of 7-acetoxycoumarin-4-acetic acid 5 (945 mg, 3.6 mmol, 1 eq) in 47 ml of

Anhydrous dichloromethane at room temperature under a nitrogen atmosphere. A solution of N, N'dicyclohexylcarbodiimide (818 mg, 4 mmol, 1.1 eq) in 10 ml of anhydrous dichloromethane was added. The reaction mixture was stirred for 15 min, after which it was diluted with dichloromethane and filtered. The filtrate was concentrated in vacuo. Column chromatography on silica gel (CH2Cl2 and then CH2Cl2: AcOEt, 10: 1) gave the product 6a (1.37 g, 96%): Rf 0.78 (CH2Cl2: AcOEt, 5: 1); 1H NMR (300 MHz, CDCl3): 5 7.61 (d, 1H, J5.6 8.7 Hz, H-5), 7.15 (d, 1H, J6.8 2.1 Hz, H- 8), 7.07 (dd, 1H, J6.8 2.3, J5.6 8.7 Hz, H-6), 6.42 (s, 1H, H-3), 4.77 (s, 2H, CH2CCl3), 3.91 (2s, 2H, CH2CO2), 2.33 (s, 3H, OAc); NMR-13C (75 MHz, CDCl3): 5 168.6, 167.0, 154.5, 153.5, 146.5, 125.5, 118.5, 117.0, 116.5, 110.9 , 74.6, 37.7, 21.2; ESI-MS: m / z 393 [M + H] +.

[0034] 7-Hydroxycoumarin-4-acetate (2 ’, 2’, 2’-trichlorethyl) (6b). A solution of 6a (1.08 g, 2.74 was prepared

65 mmol, 1 eq) in 108 ml of anhydrous tetrahydrofuran under a nitrogen atmosphere at room temperature. A solution of 2M ammonia in 2-propanol (6.8 ml, 13.7 mmol, 5 eq) was added dropwise. The reaction mixture was stirred at room temperature in a tightly sealed flask for 18 h. The reaction mixture was concentrated in vacuo. Purification by column chromatography on silica gel (CH2Cl2 and then CH2Cl2: AcOEt 10: 1 to 5: 1) provided product 6b (753 mg, 78%): Rf 0.6 (CH2Cl2: AcOEt, 5: 1) ; 1H NMR (300 MHz, DMSO-d6): 5 7.55 (d, 1H, J5.6 8.7 Hz, H-5), 6.79 (dd, 1H, J6.8 2.3, J5 , 6 8.7 Hz, H-6), 6.74 (d, 1H, J6.8 2.3 Hz, H-8), 6.31 (s, 1H, H-3), 4.94 ( s,

5H, CH2CCl3), 4.14 (2s, 2H, CH2CO2); NMR-13C (75 MHz, DMSO-d6): 5 168.4, 161.8, 160.5, 155.5, 149.2, 127.3, 113.5, 112.9, 111.5, 102 , 8, 95.5, 74.0, 36.9; ESI-MS: m / z 351 [M + H] +.

[0035] 7-O- (Methyl-2 ', 3', 4'-tri-O-acetyl-a-idopiranosyluronate) coumarin-4-acetate (2 ", 2", 2 "-trichloroethyl) (7) A suspension of 6b (703 mg, 2 mmol, 1.26 eq) and LiClO4 / SiO2 (200 mg) in 2 ml of anhydrous dichloromethane was stirred at room temperature under nitrogen atmosphere, 1.1 was added dropwise , 1,3,3,3-hexamethyldisilazane (835 µL, 4 mmol, 2.52 eq.) The reaction mixture was stirred for 35 min. The reaction mixture was diluted with dichloromethane and filtered. The filtrate was concentrated by rotavapor, providing 7-O-trimethylsilylcumarin-4-acetate of (2 ', 2', 2'trichloroethyl) 6c, which was used in the next step without further purification. 534 mg, 1.6 mmol, 1 eq) and the glycosyl acceptor previously prepared 6c in 10 ml of anhydrous dichloromethane under a nitrogen atmosphere, diethyl boron-ethyl ether trifluoride (196 µl, 1.6 was added dropwise) mmol, 1 eq), after which d He allowed the reaction mixture to warm to room temperature. The reaction flask was tightly sealed, the reaction mixture was stirred for 1.5 h and diethyl boroeterate trifluoride (88 µl) was added. After stirring for 20 min, the reaction was diluted with 200 ml of dichloromethane and washed with 100 ml of water, 100 ml of saturated sodium bicarbonate and 100 ml of brine. The organic phase was dried over MgSO4 and concentrated in vacuo with additional coevaporations with toluene. Column chromatography on silica gel (CH2Cl2 and then CH2Cl2: AcOEt 10: 1 to 5: 1) provided product 7 (934 mg, 88%): [a] D -80 ° (c1, CHCl3); Rf 0.5 (CH2Cl2: AcOEt 5: 1); 1H NMR (300 MHz, CDCl3): 5 7.53 (d, 1H, J5.6 8.7 Hz, H-5), 7.06 (d, 1H, J6.8 2.5 Hz, H- 8), 7.01 (dd, 1H, J6.8 2.5, J5.6 8.9 Hz, H-6), 6.33 (s, 1H, H-3), 5.84 (d, 1H, J1 ', 2' 2.5 Hz, H-1 '), 5.20 (m, 2H, H3', H-4 '), 5.05 (m, 1H, H-2'), 4 , 89 (m, 1H, H-5 '), 4.77 (2s, 2H, CH2CCl3), 3.87 (s, 2H, CH2CO2), 3.77 (s, 3H, CO2Me),

25 2.16-2.09 (3s, 9H, 3 OAc); NMR-13C (75 MHz, CDCl3) 5 169.5, 169.4, 169.0, 167.9, 167.2, 160.2, 158.9, 155.3, 146.7, 126.0, 115.7, 114.1, 113.2, 104.9, 95.7, 94.5, 74.6, 67.8, 67.0, 66.8, 52.9, 37.7, 20, 9, 20.9, 20.7; ESI-MS: m / z 667 [M + H] +.

[0036] 7-O- (Methyl-2 ’, 3’, 4’-tri-O-acetyl-a-L-idopyranosyluronate) coumarin-4-acetamide from (N- [4 "- (terc

30-butoxycarbonylamino) butyl]) (9a). Glycoside 7 (831 mg, 1.2 mmol, 1 eq) was dissolved in 41 ml of anhydrous tetrahydrofuran at room temperature. The solution was cooled to 0 ° C and 90% aqueous acetic acid (5.5 ml) was added. Finally, copper chloride (167 mg, 1.2 mmol, 1 eq) and zinc powder (813 mg, 12.4 mmol, 10 eq) were added. The reaction mixture was stirred at 0 ° C for a total of 39 h, during which zinc powder (813 mg, 12.4 mmol, 10 eq) was added after 15 and 25 h of reaction. The reaction mixture was filtered through Celite and the

35 filtered under vacuum. The residue was solubilized in 200 ml of dichloromethane and washed with 150 ml of water (twice) and 150 ml of brine. The organic phase was dried over MgSO4 and concentrated in vacuo. Column chromatography on silica gel (CH2Cl2 and then CH2Cl2: AcOEt 5: 1 to 2: 1; all solvents with 1% acetic acid) provided product 8 (634 mg, 95%). A solution of acid 8 (627 mg, 1.2 mmol, 1 eq) in 20 ml of anhydrous tetrahydrofuran was cooled to 0 ° C. N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (245 mg, 1.28 was added

40 mmol, 1.1 eq) and 1-hydroxybenzotriazole (196 mg, 1.28 mmol, 1.1 eq) and the suspension was stirred for 30 min at 0 ° C. A solution of N-Boc-1,4-diaminobutane (223 µL, 1.2 mmol, 1 eq) in 2 mL of anhydrous N, N-dimethylformamide was slowly added to the suspension. The reaction mixture was allowed to warm to room temperature and stirred for 3

h. The reaction mixture was concentrated in vacuo. The residue was taken up in 250 ml of ethyl acetate and washed with 150 ml of 1M HCl, 150 ml of water and 150 ml of brine. The organic phase was dried over MgSO4 and concentrated in vacuo. 45 Column chromatography on silica gel (toluene: acetone 3: 1 to 2: 1) provided product 9a (528 mg, 65%): [a] D -70 ° (c 0.85, CHCl3); Rf 0.38 (CH2Cl2: MeOH, 95: 5); 1H NMR (300 MHz, CDCl3): 5 7.67 (d, 1H, J5.6 8.7 Hz, H5), 7.03 (d, 1H, J6.8 2.3 Hz, H-8) , 6.99 (dd, 1H, J6.8 2.5, J5.6 8.7 Hz, H-6), 6.29 (s, 1H, H-3), 5.84 (d, 1H, J1 ', 2' 2.1 Hz, H1), 5.20 (m, 2H, H-3 ', H-4'), 5.04 (m, 1H, H-2 '), 4.89 ( d, 1H, J4 ', 5' 2.1 Hz, H-5 '), 3.77 (s, 3H, CO2Me), 3.65 (s, 2H, CH2CONH), 3.26 (m, 2H, CH2NHCO), 3.09 (m, 2H, CH2NHCO), 2.17-2.09 (3s, 9H, 3 OAc), 1.49 (m, 4H, CH2-CH2),

50 1.42 (s, 9H, CMe3); NMR-13C (75 MHz, CDCl3) 5 169.4, 169.4, 169.0, 167.8, 167.6, 160.7, 158.8, 156.4, 155.2, 149.7, 126.7, 114.5, 114.5, 113.2, 104.5, 95.6, 79.4, 67.7, 66.9, 66.6, 52.8, 39.8, 28, 5, 20.8, 20.8, 20.6; ESI-MS: m / z 707 [M + H] +.

[0037] 7-O- (a-L-idopyranosyluronate) coumarin-4-acetamide of (N- [4 "- (tert-butoxycarbonylamino) butyl]) (9b).

A solution of 9a (98 mg, 0.165 mmol, 1 eq) in 16 ml of methanol was cooled to 0 ° C. A solution of 0.5 M sodium methoxide in methanol (140 µL, 0.07 mmol, 0.4 eq) was added dropwise. The reaction mixture was stirred at 0 ° C for 1.5 h. The reaction mixture was neutralized with Amberlite IR-120 (H +) and filtered. The filtrate was concentrated in vacuo. Column chromatography on silica gel (CH2Cl2 and then CH2Cl2: MeOH 9: 1) provided product 9b (69 mg, 86%): 1 H-NMR (300 MHz, CD3OD): 7.69 (d, 1H, J5.6 9.7 Hz, H-5), 7.14 (d, 1H, J6.8 2.3 Hz, H

60 8), 7.13 (dd, 1H, H-6), 6.28 (s, 1H, H-3), 5.76 (d, 1H, J1 ', 2' 3.9 Hz, H- 1 '), 4.75 (d, 1H, J4', 5 '3.5 Hz, H-5'), 3.97-3.89 (m, 2H, H-2 ', H-3') , 3.77 (s, 3H, CO2Me), 3.74 (s, 2H, CH2CONH), 3.73 (m, 1H, H-4 '), 3.21 (m, 2H, CH2NHCO), 3, 03 (m, 2H, CH2NHCO), 1.49 (m, 4H, CH2-CH2), 1.42 (s, 9H, CMe3); ESI-MS: m / z 581 [M + H] +.

Example 2

65 Preparation of 2-sulfated α-L-iduronate glycoside 11

[0038] The preparation of the 2-sulfated α-L-iduronate 11 glycoside is described below and is illustrated in FIGURE 1.

[0039] Synthesis of aL-iduronate 10 glycoside methyl ester. Starting material 9b (164.5 mg, 0.28 mmol, 1 eq), prepared as described in example 1, was solubilized in 16 ml of anhydrous methanol and dibutyltin (IV) oxide (106 mg, 0.42 mmol, 1.5 eq, Aldrich) was added. The reaction mixture was heated at reflux for 40 minutes, after which the dibutyltin oxide was completely dissolved. The mixture was allowed to cool

10 reaction and concentrated in vacuo. The residue was coevaporated once to remove traces of water.

[0040] The residue was solubilized in 16 ml of anhydrous N, N-dimethylformamide. Sulfur trioxide trimethylamine complex (59.1 mg, 0.42 mmol, 1.5 eq, Aldrich) was added and the reaction mixture was heated at 55 ° C for 24 hours. The reaction mixture was allowed to cool and then quenched with methanol, before concentrating in vacuo. To convert the trimethylammonium salt product into sodium salt, the residue was subjected to cation exchange chromatography [Dowex 50WX8-400 (Na +), 1 x 4 cm] using methanol as eluent. The sodium salt was purified by column chromatography on silica using methanol / chloroform / water (5/8/1) to give the glycoside methyl ester of aL-iduronate 10. TLC (silica, methanol / chloroform / water 5/8 / 1): Rf = 0.6. 1H NMR (300 MHz, CD3OD): 1.43 (s, 9H, tert-butyl); 1.50 (m, 4H, CH2CH2); 3.04 (m, 2H, CH2N); 3.21 (t, 2H, CH2N); 3.74 (s a, 2H, CH2CO); 3.76 (s, 3H,

20 CO2Me); 3.99 (t a, 1H, H-4); 4.19 (t a, 1 H, H-3); 4.50 (m, 1H, H-2); 4.81 (d, 1 H, H-5); 6.00 (s a, 1H, H-1); 6.28 (s, 1 H, coumarinvinyl CH); 7.16-7.19 (m, 2H, CH coumarin); 7.70 (d, 1H, CH coumarin).

[0041] Synthesis of 2-sulfated α-L-iduronate 11 glycoside. Compound 10 was solubilized in 15.4 ml of methanol / water (1/1) at room temperature. An aqueous solution of 0.1 M sodium hydroxide was added in 25 increments of 0.1 eq of NaOH (283 µl, 0.03 mmol) until the pH of the solution reached approximately 8 (pH paper). The pH was maintained by incremental additions of the 0.1 M NaOH solution as the reaction proceeded (every 15-30 minutes). The reaction mixture was stirred for 5.5 h (1.3 eq. Of NaOH added), after which it was concentrated in vacuo to remove methanol and finally lyophilized overnight. The residue was purified by column chromatography on silica using methanol / chloroform / water (5/8/1), to give the glycoside of a30 L-iduronate 2-sulfated 11 (96% 2-sulfated, 4% 4-sulfated ) with 61% overall yield from compound 9b. TLC (silica, methanol / chloroform / water, 5/8/1): Rf = 0.2. 1H NMR (300 MHz, CD3OD): 1.43. (s, 9H, tertbutyl); 1.50 (m, 4H, CH2CH2); 3.04 (t, 2H, CH2N); 3.21 (t, 2H, CH2N); 4.07 (s a, 1 H, H-4); 4.17 (s a, 1H, H-3); 4.48 (s a, 1 H, H-2); H-5 under the peak of the water; 6.14 (s a, 01H, H-1); 6.17 (s, 1 H, coumarinvinyl CH); 7.07-7.12 (m, 2H, coumarin CH); 7.53 (d, 1H, CH coumarin). Mass spectrometry with electrospray ionization:

35 (negative mode) (M-H) -1, calculated 645.2, observed 645.3.

[0042] Although illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the scope of the invention.

Claims (6)

1. A method for sulfating an α-L-iduronate glycoside in position 2, comprising: 5 a) treating an α-L-iduronate glycoside having hydroxyl groups in positions 2 and 4 in cis relation with a tin reagent, providing a 2,4-stanylene acetal of α-L-iduronate glycoside; Y b) treating the 2,4-stanylene acetal with a sulphating agent, providing a glucoside of a-L-iduronate 2sulfated. 10 2. The method of claim 1, wherein the 2-sulfated glycoside is sulphated in position 2 with a selectivity greater than 90%, preferably wherein the 2-sulfated glycoside is sulfated in position 2 with a selectivity greater than 95 %. 3. The method of claim 1, wherein the tin reagent is a dialkyltin oxide (VI) or in which the tin reagent is dibutyltin oxide (IV). 4. The method of claim 1, wherein the sulfating agent is sulfur trioxide; preferably wherein the sulfating agent is a sulfur trioxide complex selected from the group 20 consisting of sulfur trioxide and trimethylamine complex, sulfur trioxide and pyridine complex and sulfur trioxide complex and N, N-dimethylformamide.

5.

The method of claim 1, wherein the iduronate glycoside has the formula:

6.

The method of claim 1, wherein the 2-sulfated iduronate glycoside has the formula:

7.

The process of claim 6, further comprising saponifying the 2-sulfated iduronate glycoside methyl ester, providing a 2-sulfated iduronate glycoside having the formula: